| Home | E-Submission | Sitemap | Contact us |  
Korean J Parasitol > Volume 30(3):1992 > Article

Original Article
Korean J Parasitol. 1992 Sep;30(3):191-199. English.
Published online Mar 20, 1994.  http://dx.doi.org/10.3347/kjp.1992.30.3.191
Copyright © 1992 by The Korean Society for Parasitology
Isolation and partial characterization of cysteine proteinase from sparganum
C Y Song,D H Choi,T S Kim and S H Lee*
Department of Biology, Chung-Ang University, Seoul 156-756, Korea.
*Department of Parasitology, Seoul National University College of Medicine, Seoul 110-460, Korea.

A proteolytic enzyme was purified from the tissue extract of spargana (plerocercoids of Spirometra erinacei) by DEAE-Trisacryl M ion exchange chromatography and thiopropyl-sepharose affinity chromatography resulted in a 21-fold purification. The proteinase activity was assayed with a synthetic fluorescent substrate, carbobenzoxy-phenylalanyl-7-amino-4-trifluoromethyl-coumarin. SDS-polyacrylamide gel electrophoresis of the purified materials revealed a single 28,000 dalton band. Inhibitor profiles of the band indicated that it belonged to cysteine endopeptidases. It exhibited identical pH curves with optimum at pH 5.5, and 50% activity from pH 4.7 to 8. It could completely degrade collagen chains to three identical products. It also showed some activity on hemoglobin. Furthermore, the band on immunoblots was reactive to the sera of sparganosis patients. These results suggest that the proteolytic enzyme belongs to cysteine proteinase which plays a role in the tissue penetration. Also it may be used as the antigen for diagnosis of active sparganosis.


Fig. 1
Affinity chromatography with thiolpropyl-sepharose 4B. The supernatants were loaded onto DEAE-Trisacryl M chromatography, pooled, dialyzed with Sephadex G-25 and concentrated. The active peaks were loaded onto thiolpropyl-sepharose 4B, 0.1M Tris-HCl containing 0.3 NaCl, 1mM EDTA buffer (pH 7.5) and 2.0ml fractions (Fno) were assayed for activity on CBZ-phe-arg-AFC (○). Fractions were monitored for protein content (•) at 280 nm.

Fig. 2
10~20% gradient SDS-PAGE of purification steps of plerocercoid extracts. Lane A, active peak from thiolpropyl-sepharose 4B; Lane B, active peak from DEAE-Trisacryl M; Lane C, extracts; Lane D, molecular weight protein markers; phosphorylase B (94 kDa), albumin (67 kDa), ovalbumin (43kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa), α-lactalbumin (14kDa).

Fig. 3
Estimation of molecular weight of proteinases from Spirometra plerocercoid by AcA54 gel filtration(A) and SDS-PAGE(B).

Fig. 4
Effect of pH on cysteine proteinase activity from plerocercoid extracts.

Fig. 5
Degradation of collagen (upper) and hemoglobin (lower) by proteinases from plerocercoid extracts. Upper; Lane A, molecular weight protein marker; Lane B, collagen control; Lane C, 30 min; Lane D, 1hr. Lower; Lane A, molecular weight protein markers; Lane B, hemoglobin control; Lane C, 2hr; Lane D, 4hr.

Fig. 6
IgG reactivity to purified plerocercoid cysteine proteinase(▴) from patients with sparganosis. Lane A, molecular weight protein marker; Lane B, C, D, E and F, human sera of sparganosis; Lane G, normal human serum.


Table 1
Purification of cysteine proteinase from solube extracts of sparganum

Table 2
Effect of endopeptidase inhibitors on proteinase purified from sparganum extracts

1. Chappell CL, Dresden MH. Schistosoma mansoni: proteinase activity of "hemoglobinase" from the digestive tract of adult worms. Exp Parasitol 1986;61(2):160–167.
2. Chappell CL, Dresden MH. Purification of cysteine proteinases from adult Schistosoma mansoni. Arch Biochem Biophys 1987;256(2):560–568.
3. Chappell CL, Dresden MH. Antibody response to a purified parasite proteinase (SMw32) in Schistosoma mansoni infected mice. Am J Trop Med Hyg 1988;39(1):66–73.
4. Chi JG, Chi HS, Lee SH. Histopathologic Study On Human Sparganosis. Korean J Parasitol 1980;18(1):15–23.
5. Cho SY, Kang SY, Kong Y. Purification of antigenic protein of sparganum by immunoaffinity chromatography using a monoclonal antibody. Korean J Parasitol 1990;28(3):135–142.
6. Choi SH, Kang SY, Kong Y, Cho SY. Antigenic protein fractions reacting with sera of sparganosis patients. Korean J Parasitol 1988;26(3):163–167.
7. Dresden MH, Asch HL. Proteolytic enzymes in extracts of Schistosoma mansoni cercariae. Biochim Biophys Acta 1972;289(2):378–384.
8. Fukase T, et al. Jpn J Parasitol 1985;34:351–360.
9. Fung CF, Ng TH, Wong WT. Sparganosis of the spinal cord. Case report. J Neurosurg 1989;71(2):290–292.
10. Holodniy M, Almenoff J, Loutit J, Steinberg GK. Cerebral sparganosis: case report and review. Rev Infect Dis 1991;13(1):155–159.
11. Hong ST, Kim KJ, Huh S, Lee YS, Chai JY, Lee SH, Lee YS. The changes of histopathology and serum anti-sparganum IgG in experimental sparganosis of mice. Korean J Parasitol 1989;27(4):261–269.
12. Ishii A. Jpn J Parasitol 1973;22:75–78.
13. Kim CH, Choi WS. [Immunohistochemical observation on the antigens inducing IgG and IgM antibodies against sparganum]. Korean J Parasitol 1991;29(4):339–353.
14. Kim H, Kim SI, Cho SY. Serological Diagnosis Of Human Sparganosis By Means Of Micro-ELISA. Korean J Parasitol 1984;22(2):222–228.
15. Kim LS, Kong Y, Kang SY, Cho SY. Immunohistochemical localization of 36 and 29 kDa proteins in sparganum. Korean J Parasitol 1992;30(1):25–31.
16. Kron MA, Guderian R, Guevara A, Hidalgo A. Abdominal sparganosis in Ecuador: a case report. Am J Trop Med Hyg 1991;44(2):146–150.
17. Kwa BH. Studies on the sparganum of Spirometra erinacei. II. Proteolytic enzymes(s) in the scolex. Int J Parasitol 1972;2(1):29–33.
18. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680–685.
19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1):265–275.
20. McKerrow JH, Pino-Heiss S, Lindquist R, Werb Z. Purification and characterization of an elastinolytic proteinase secreted by cercariae of Schistosoma mansoni. J Biol Chem 1985;260(6):3703–3707.
21. Nakamura T, et al. Jpn J Parasitol 1982;31 Suppl:8.
22. Nakamura T, et al. Jpn J Parasitol 1982;31 Suppl:36.
23. Nakamura T, et al. Jpn J Parasitol 1984;33 Suppl:24.
24. Okabe H, et al. Kurume J Med 1957;20:907–913.
25. Osaki Y. Ultrastructural studies on the plerocercoid of Spirometra erinacei in experimental sparganosis. Parasitol Res 1990;76(6):466–472.
26. Rege AA, Dresden MH. Strongyloides spp.: demonstration and partial characterization of acidic collagenolytic activity from infective larvae. Exp Parasitol 1987;64(3):275–280.
27. Ruppel A, Rother U, Vongerichten H, Lucius R, Diesfeld HJ. Schistosoma mansoni: immunoblot analysis of adult worm proteins. Exp Parasitol 1985;60(2):195–206.
28. Sarma DP, Weilbaecher TG. Human sparganosis. J Am Acad Dermatol 1986;15(5 Pt 2):1145–1148.
29. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76(9):4350–4354.
30. Zerda KS, Dresden MH, Chappell CL. Schistosoma mansoni: expression and role of cysteine proteinases in developing schistosomula. Exp Parasitol 1988;67(2):238–246.
Editorial Office
c/o Department of Medical Environmental Biology
Chung-AngUniversity College of Medicine, Dongjak-gu, Seoul 06974, Korea
Tel: +82-2-820-5683   Fax: +82-2-826-1123   E-mail: kjp.editor@gmail.com
About |  Browse Articles |  Current Issue |  For Authors and Reviewers
Copyright © 2019 by The Korean Society for Parasitology and Tropical Medicine. All rights reserved.     powerd by m2community